Few publications describe a method for removing endotoxins by precipitation with alcohols, since endotoxins and DNA behave very similarly. Using currently established techniques, alcohols such as ethanol and isopropanol cause DNA and endotoxins to precipitate out together.
In the process described in WO95/21178A1 isopropanol is substituted for ethanol as the fluid phase in anion exchange chromatography. Substitution of the univalent alcohol ethanol with the similarly univalent alcohol isopropanol leads to an improved transfection efficiency.
WO 99/063076 A1 describes the removal of endotoxins using ethanol as the fluid phase in anion exchange chromatography. In addition, it describes the use of Triton® X-114, which is known to be well-suited to the removal of endotoxins in this application.
EP 0 407037B1 describes a purification process that encompasses a complicated and onerous fractionated separation with ethanol. Deoxycholate is additionally used as a frequently described and recognized detergent for the removal of endotoxin. The process described is said to separate endotoxins from bacterial polysaccharides, but nucleic acid purification is neither the goal nor the theme of this process.
According to WO2005/003152A1, protein complexes can be separated from endotoxins after binding to a chromatographic surface by means of alkane diols. This process does not function to purify plasmid DNA since it requires a cation exchanger. This limitation applies generally to the direct utility of ion exchange eluates.
The use of basic compounds as helpers for chromatographic purification is recognized in the literature.
U.S. Pat. No. 6,699,386 B2 describes the use of a basic material in the affinity chromatographic separation of endotoxins, wherein the basic material is present bound to a solid surface.
The production of the corresponding purification column is laborious and requires additional purification steps to separate the eluted, endotoxin-free substances. In addition, the capacity of an affinity chromatographic column is limited and dependent upon the quantity of material used, which unnecessarily increases the dead volume and thereby the eluate volume.
U.S. Pat. No. 7,714,111 B2 describes the use of a washing buffer containing an arginine derivative to remove impurities. The process presented in the article is proposed for purification of a monoclonal antibody, which is bound by its Fc-portion to a protein-A affinity chromatography column. A heterologous group of substances can be separated by the aforementioned antibody. This includes both endotoxins and nucleic acids. This process can therefore not be used for the purification of nucleic acids.
From WO 2005/111059 A2, a process for the separation of endotoxins from samples containing plasmid-DNA is disclosed, which utilizes a carbohydrate-based non-ionic detergent together with a silica carrier material. In this process, it can be viewed as a disadvantage that use of the common silica membrane as a carrier material can lead to blockage, particularly when larger quantities of nucleic acids are to be separated. In addition, the yields of nucleic acids are not always satisfactory and the separation performance is equally unsatisfactory for endotoxins.